Mitogenomic phylogeny of nymphalid subfamilies confirms the basal clade position of Danainae (Insecta: Lepidoptera: Nymphalidae)

Abstract The phylogenetic relationships among the nymphalid subfamilies have largely been resolved using both morphological and molecular datasets, with the exception of a conflicting basal clade position for Libytheinae or Danainae that remains contentious between morphological and molecular studies. Several phylogenomic analyses have found that the danaine clade is sister to other nymphalid subfamilies; however, it largely depends on utilizing different molecular datasets, analysis methods, and taxon sampling. This study aimed to resolve the basal clade position and relationships among subfamilies and tribes of Nymphalinae by combining the most comprehensive available mitogenomic datasets with various analyses methods by incorporating a new Symbrenthia lilaea Hewitson sequence data. Phylogenetic relationships among 11 nymphalid subfamilies and the tribes of Nymphalinae were inferred by combining new and available mitogenomic sequence data from 80 ingroup and six outgroup species. The phylogenetic trees were reconstructed using maximum‐likelihood (ML) and Bayesian inference (BI) methods based on five concatenated datasets: amino acid sequences and nucleotides from different combinations of protein‐coding genes (PCGs), ribosomal RNA (rRNAs), and transfer RNA (tRNAs). Danainae is well‐supported as the basal clade and sister to the remaining nymphalid subfamilies, except for the paraphyletic Libytheinae. Libytheinae was either recovered as a sister to the danaine clade followed by the satyrine clade or sister to the nymphaline + heliconiine clades, and is consistent with recent phylogenetic studies on Nymphalidae. The monophyletic Nymphalinae has been recovered in all analyses and resolves tribal‐level relationships with high support values in both BI and ML analyses. We supported the monophyletic Nymphalini as a sister clade to Victorini, Melitaeini, and Kallimini + Junoniini with high supporting values in BI and ML analyses, which is consistent with previously published morphological and molecular studies.

tious between morphological and molecular studies. Several phylogenomic analyses have found that the danaine clade is sister to other nymphalid subfamilies; however, it largely depends on utilizing different molecular datasets, analysis methods, and taxon sampling. This study aimed to resolve the basal clade position and relationships among subfamilies and tribes of Nymphalinae by combining the most comprehensive available mitogenomic datasets with various analyses methods by incorporating a new Symbrenthia lilaea Hewitson sequence data. Phylogenetic relationships among 11 nymphalid subfamilies and the tribes of Nymphalinae were inferred by combining new and available mitogenomic sequence data from 80 ingroup and six outgroup species. The phylogenetic trees were reconstructed using maximum-likelihood (ML) and Bayesian inference (BI) methods based on five concatenated datasets: amino acid sequences and nucleotides from different combinations of protein-coding genes (PCGs), ribosomal RNA (rRNAs), and transfer RNA (tRNAs). Danainae is well-supported as the basal clade and sister to the remaining nymphalid subfamilies, except for the paraphyletic Libytheinae. Libytheinae was either recovered as a sister to the danaine clade followed by the satyrine clade or sister to the nymphaline + heliconiine clades, and is consistent with recent phylogenetic studies on Nymphalidae. The monophyletic Nymphalinae has been recovered in all analyses and resolves tribal-level relationships with high support values in both BI and ML analyses. We supported the monophyletic Nymphalini as a sister clade to Victorini, Melitaeini, and Kallimini + Junoniini with high supporting values in BI and ML analyses, which is consistent with previously published morphological and molecular studies.

K E Y W O R D S
brush-footed butterflies, common jesters, mitochondrial genome, Papilionoidea, systematics

| INTRODUC TI ON
Nymphalidae is the largest butterfly family with 12 extant subfamilies, 40 tribes, 559 genera, and ~6400 described species distributed worldwide: Danainae, Calinaginae, Charaxinae, Satyrinae, Libytheinae, Pseudergolinae, Apaturinae, Biblidinae, Cyrestinae, Nymphalinae, Heliconiinae, and Limenitidinae (Chazot et al., 2021;Wahlberg et al., 2009). Nymphalid butterflies, which likely originated in the Oriental region and repeatedly dispersed throughout the rest of the world during the early Eocene, diverged from its sister clade around 90 mya ago in the Late Cretaceous, and started to diversify around 84.6 mya ago (Chazot et al., 2021). The taxonomic levels and phylogenetic relationships among different subfamilies have mainly been resolved based on both morphological and molecular datasets. However, the basal clade position is still unresolved: either Danainae based on molecular datasets (Chazot et al., 2021;Hao et al., 2013;Shi et al., 2015;Wu et al., 2014), or Libytheinae (Espeland et al., 2018;Freitas & Brown, 2004;Wahlberg et al., 2003Wahlberg et al., , 2009) based on molecular datasets, adult morphology, and similarities in the immatures with Pieridae, a hypothesis proposed 65 years ago (previously treated as distinct family; Ehrlich, 1958), has been suggested as the sister to all Nymphalidae. However, recent mitogenomic and morphological studies support Danainae as the basal clade of Nymphalidae, whereas the paraphyletic Libytheinae remains a mystery Shi et al., 2015;Wu et al., 2014). Kawahara (2009) published the landmark contribution on the cladistic analyses of both extant and extinct species of Libytheinae to date, but the complete mitochondrial genome data of libytheine species are scarce and must be updated to understand its paraphyletic relationship among other nymphalid subfamilies.
The subfamily Nymphalinae comprises five tribes, Nymphalini, Kallimini, Victorinini, Junoniini, Melitaeini, and probably the Coeini (Su et al., 2017;Wahlberg et al., 2005). Nymphalinae has been recovered as a monophyletic clade and strongly supported in both morphological and mitogenomic studies (Shi et al., 2015;Su et al., 2017;Wahlberg et al., 2003Wahlberg et al., , 2005Wahlberg & Wheat, 2008;Wu et al., 2014); however, the tribe-level relationships are mainly dependent on the use of different molecular datasets, analyses methods, and taxon sampling (Nylin et al., 2001;Su et al., 2017;Wu et al., 2014). Wahlberg et al. (2005) supported the monophyletic Nymphalinae with Nymphalini as a basal clade and sister to Victorini, Melitaeini, and Kallimini + Junoniini based on both morphological and molecular datasets. However, the Kallimini + Junoniini clade does not support recent mitogenomic studies, which either recovered Kallimini as sister to Junoniini + Melitaeini (Wu et al., 2014) or sister to the clade comprising Melitaeini and Junoniini (Shi et al., 2015), or paraphyletic Kallimini (Kim et al., 2021). These findings might be due to taxon sampling biases; however, the mitogenomic data on Nymphalidae has significantly increased in the last two decades and are available on NCBI.
This study aims to reconstruct the phylogenetic relationships among the nymphalid subfamilies and tribes of Nymphalinae by using the most comprehensive mitogenomic datasets available on Nymphalidae. Here, we included 80 ingroup species in 11 subfamilies that recovered consistent topologies and supported Danainae as a basal clade to all subfamilies. The subfamily Nymphalinae also appeared as monophyletic and Nymphalini as a basal clade and is sister to Victorini, Melitaeini, and Kallimini + Junoniini.

| Taxon sampling, DNA extraction, and sequencing
The Oriental nymphalid butterfly genus Symbrenthia Hübner, 1819 (Nymphalinae), commonly called jesters, belongs to the tribe Nymphalini which has 14 recognized species (Fric et al., 2004(Fric et al., , 2022Kunte, 2010), mainly distributed in the Oriental region and reaching New Guinea and the eastern Palaearctic (Bozano & Floriani, 2012). The complete mitogenome was sequenced using high-throughput sequencing on the Illumina Novaseq 6000 platform with an average insert size of 350 bp and a paired-end 150 bp (PE 150) sequencing strategy to generate a sequencing data not less than 2 GB. Raw reads were trimmed of adapters using Trimmomatic (Bolger et al., 2014).

| Phylogenetic analyses
In order to reconstruct the phylogenetic relationships among the nymphalid subfamilies and tribes of Nymphalinae, we retrieved the complete mitochondrial genome sequences of 80 species from 11 subfamilies available on NCBI, including the new sequence data of S. lilaea as ingroup, and six outgroup species from the closely related families Lycaenidae (three species), and Pieridae (three species). The GenBank accession numbers, mitogenome size, locality data, and references for the species studies are presented in Table 1. The complete mitogenome genes were extracted using PhyloSuite (v1.2.2; Zhang et al., 2020). The nucleotide sequences of 13 individual PCGs were aligned using MUSCLE (Edgar, 2004) in MEGA 7.0 (Kumar et al., 2016), and the stop codons were manually deleted. Alignments of 13 PCGs were concatenated using FASconCAT_v1.0 (Kück & Meusemann, 2010). The amino acid sequences of 13 PCGs were aligned using MUSCLE in MEGA 7.0. RNA gene alignment was conducted using MAFFT and then trimmed using trimAl to align the sequence. Finally, we concatenated the aligned sequenced of each gene to generate five datasets using PhyloSuite: (1)  Phylogenetic trees were reconstructed using the ML and BI methods. ML analyses were performed in IQ-TREE (Nguyen et al., 2015) using ultrafast bootstrap with 5000 replicates as implemented on the webserver (http://iqtree.cibiv.univie.ac.at/), and the BI analyses were performed in MrBayes v.3.2.6 (Ronquist et al., 2012) implemented in PhyloSuite software with various data partition schemes and best-fitting models determined by PartitionFinder (Lanfear et al., 2017; Appendix S2, Tables S1-S10). The BI analyses contains four simultaneous Markov chain Monte Carlo (MCMC) runs of 2 million generations, and sampled every 1000 generations. The initial 25% of the sampled data were discarded as burn-in. Other parameters were kept at default settings. The phylogenetic trees were visualized and edited in the Interactive Tree of Life (iTOL: https://itol. embl.de) version 5 (Letunic & Bork, 2021).

| Genome structure and nucleotide composition
The complete mitochondrial genome of S. lilaea is sequenced, annotated, and analyzed in this study. The complete mitochondrial genome of S. lilaea is 15,726 bp in length, containing 37 genes (22 F I G U R E 1 Symbrenthia lilaea. (a, b) Live adult, dorsal and lateral habitus; (c, d) Dorsal and ventral habitus. © Lan Zhang.
transfer RNA genes, two ribosomal RNA genes, and 13 proteinencoding genes) and the control region (Table 2), with the same ancestral gene order and arrangement in Nymphalidae. The circular mitogenome map of S. lilaea is shown in Figure 3. The overall nucleotide composition in S. lilaea is 87.7% versus 12.3% of A + T content and G + C content, respectively (A = 35.7%, T = 52.0%, C = 6.4%, G = 5.9%). The A + T content of isolated PCGs, tRNAs, rRNAs, and control region were all above 77% (Table 3).

| Phylogenetic analyses
We discussed the basal clade position in Nymphalidae and tribal-  In the cladistic analyses, host plants, one mitochondrial (COI), and one or two nuclear genes (EF-1α and wingless), Libytheinae has always been proposed as a basal clade to all Nymphalidae based on features from the immature stage (lacks horns or spines) and adult female characters (foreleg with tarsal claws; Brower, 2000;Freitas & Brown, 2004;Wahlberg et al., 2003). However, Shi et al. (2015) proposed Danainae as the basal group to all nymphalid subfamilies based on adult wing character (connected medius one vein and radius vein) and Libytheinae as the sister to the nymphaline + heliconiine clade based on the slightly closed or open discal cell of the forewing. This hypothesis is supported in various molecular phylogenetic studies based on both nuclear and mitochondrial genomes, which supported the danaine clade as the basal group and the uncertain phylogenetic relationship of Libytheinae among the subfamilies (Chazot et al., 2021;Hao et al., 2013;Shi et al., 2015;Wu et al., 2014).  BI, 1.00-0.81; Shi et al., 2015). rRNAs +22 tRNAs; (5) PCG123_AA (Appendix S1, Figures S1-S8). The monophyletic Nymphalinae has been recovered in several morphological and molecular studies (Kim et al., 2021;Shi et al., 2015;Su et al., 2017;Wahlberg et al., 2003Wahlberg et al., , 2005 (Su et al., 2017;Wu et al., 2014). We supported the monophyletic Nymphalini as a sister clade to Victorini, Melitaeini, and Kallimini + Junoniini with high support in BI and ML analyses using five different datasets (Appendix S1, Figures S1-S8), which is consistent with previously published morphological and molecular studies (Wahlberg et al., 2005). However, our present analyses do not support for the Kallimini to be a sister to Junoniini + Melitaeini (Wu et al., 2014), or sister to the clade comprising Melitaeini and Junoniini (Shi et al., 2015).

| CON CLUS IONS
The phylogenetic relationships among the nymphalid subfamilies and tribes of Nymphalinae were reconstructed by using five different datasets of the complete mitochondrial genome through the ML and BI methods. Here, we included 80 ingroup species in 11 subfamilies that recovered consistent topologies and supported Danainae as a basal clade to all subfamilies. Libytheinae is either recovered as a sister to the danaine clade followed by the satyrine clade or sister to the nymphaline + heliconiine clades in various analyses by using five different datasets: (1) PCG123 + 2 rRNAs + 22 tRNAs; (2) PCG123 + 2 rRNAs; (3) 13 PCGs; (4) PCG12 + 2 rRNAs + 22 tRNAs; (5) PCG123_AA. The subfamily Nymphalinae also appeared as monophyletic and Nymphalini as a basal clade and is sister to Victorini, Melitaeini, and Kallimini + Junoniini.

ACK N OWLED G M ENTS
The first author is grateful to Feng-E Li (Guizhou University, Guiyang, China), Zhu Wenli (Guizhou University, Guiyang, China), and Yuezheng Tu (China Agricultural University, Beijing, China) for helping in reconstructing the phylogenetic trees by using different datasets of the complete mitochondrial genome of Nymphalidae.  (Grant no. 2019HJ2096001006).

CO N FLI C T O F I NTER E S T S TATEM ENT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The mitogenome sequences of Symbrenthia lilaea has been deposited on GenBank and assigned accession number (OR161883) is provided.